Systemic dimorphic fungi collectively cause over one million new infections every year. Latent infections worldwide grow to the tens of millions, making them a priority for today's research. Penicillium marneffei (Pm) is of particular concern due to a marked increase in the number of cases of penicilliosis in the last 20 years, which has been concurrent with the rise in immunosuppression due to the global spread of HIV infections. Pm grows as hypha in its environmental reservoir and as yeast in mammalian hosts. The phase transition between the two growth forms is considered to be essential for its pathogenicity and the transmission of penicilliosis. The transition between hypha and yeast is reversible and can be triggered at a sharp threshold temperature of 37C. The precise mechanisms underlying the thermosensing and the phase transition in Pm remain unknown. Therefore, there is a critical need to identify key genes and regulatory processes involved in the morphogenetic control in Pm. The applicants' recent findings provide a promising, new opportunity for target identification. Using an innovative, serial culture-based experimental evolution (EE) technique, the applicants derived mutant Pm strains that undergo the hypha-to-yeast phase transition at 30C rather than 37C. DNA- and RNA- sequencing of the mutant and wild-type strains revealed important roles of a family of MADS-box transcription factor (TF) genes, especially madsB and madsA, in regulating thermal dimorphism in Pm. The expression of madsB in wild type is up-regulated 1,500-fold during the hypha-to-yeast transition. The madsB loss-of-function mutation caused by genomic deletion in mutant strains seems to be responsible for the lower threshold temperature of phase transition. Significantly, EE-derived mutants are found to be completely avirulent in the mouse model, suggesting that the precise threshold temperature at 37C for the phase transition is essential for Pm to infect and/or adapt to the host condition. Furthermore, the overexpression of madsA causes Pm to grow as hypha instead of yeast at 37C. These preliminary data led to the central hypothesis that the Pm thermosensing systems act through temperature-responsive activities of the MADS-box TFs. Based on these preliminary results, the applicants propose the following two Specific Aims: (1) determine the roles of madsB and madsA in regulating thermal dimorphism in Pm, and (2) identify the downstream targets of MadsB and MadsA. Upon completion of the proposed research, the applicants expect to critically test the functions of the two MADS-box TFs in regulating dimorphic development in Pm (Aim 1). The applicants will identify the downstream targets of MadsB and/or MadsA, and examine the functions of selected targets in Pm (Aim 2). Together, these results will provide much-needed entry points to further investigate the otherwise mysterious mechanisms underlying thermal dimorphism in this important but understudied fungal pathogen.